U.S. patent application number 10/498617 was filed with the patent office on 2005-01-27 for device for the simultaneous dialysis of a number of fluid samples.
Invention is credited to Bublitz, Renate, Horn, Anton, Kreusch, Stefan, Sammler, Gunther, Schwedler, Sina.
Application Number | 20050019774 10/498617 |
Document ID | / |
Family ID | 7708904 |
Filed Date | 2005-01-27 |
United States Patent
Application |
20050019774 |
Kind Code |
A1 |
Horn, Anton ; et
al. |
January 27, 2005 |
Device for the simultaneous dialysis of a number of fluid
samples
Abstract
The invention elates to a device for the simultaneous dialysis
of a number of fluid samples, comprising a vessel for the dialysis
fluid, with an inlet and outlet, a fill-level regulation and a
sample plate (3) with a number of similar sample vessels (5) of
.mu.l range size, arranged in a raster (n.times.8.times.12), the
upper ends of which are open and the lower ends of which are sealed
by a semi-permeable membrane (7) lying in a plane. In the raster
region of the sample vessels (5), the sample plate has no regions
or elements which extend beyond the plane of the membrane (7), or
which form or support gas barriers after dipping in the dialysis
fluid (2). The sample plate (3) comprises elements in the boundary
region thereof for the escape of air after dipping in the dialysis
fluid. Both sample fluids and dialysis fluids are agitated.
Inventors: |
Horn, Anton; (Jena, DE)
; Kreusch, Stefan; (Golmsdorf, DE) ; Sammler,
Gunther; (Jena, DE) ; Bublitz, Renate; (Jena,
DE) ; Schwedler, Sina; (Eisenach, DE) |
Correspondence
Address: |
TESTA, HURWITZ & THIBEAULT, LLP
HIGH STREET TOWER
125 HIGH STREET
BOSTON
MA
02110
US
|
Family ID: |
7708904 |
Appl. No.: |
10/498617 |
Filed: |
June 10, 2004 |
PCT Filed: |
December 10, 2002 |
PCT NO: |
PCT/DE02/04553 |
Current U.S.
Class: |
435/6.11 ;
435/287.2 |
Current CPC
Class: |
B01L 3/50255 20130101;
B01L 2400/0409 20130101; B01D 61/30 20130101; B01L 2400/0487
20130101; G01N 2001/4016 20130101; B01L 3/5025 20130101; B01D 61/28
20130101; B01L 2300/0877 20130101; B01L 9/523 20130101; G01N 1/34
20130101 |
Class at
Publication: |
435/006 ;
435/287.2 |
International
Class: |
C12Q 001/68; C12M
001/34 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 10, 2001 |
DE |
101609752 |
Claims
1-35. (Cancelled)
36. An apparatus for simultaneous dialysis of a multiplicity of
liquid samples, the apparatus comprising: a dialysis container
adapted for receiving a dialysate therein, means for inflow and
outflow of the dialysate, and a level controller; at least one
sample plate disposable in the container for contact with the
dialysate, the sample plate including a multiplicity of equal
sample wells with a (n.times.8.times.12) matrix arrangement,
wherein the wells define holding capacities for microliter volumes,
open upper ends, and lower ends closed by a semipermeable dialysis
membrane lying in a plane, whereby the sample plate does not
protrude beyond the plane of the semipermeable membrane in an area
defined by the matrix arrangement, the sample plate further
including means for releasing air imprisoned between the sample
plate and the dialysate; means for holding the sample plate in
contact with a surface of the dialysate; and means for moving at
least one of the sample plate and the dialysate.
37. The apparatus of claim 36, wherein the sample wells are
substantially cylindrical.
38. The apparatus of claim 36, wherein the sample plate comprises a
geometry of conventional microtiter plates.
39. The apparatus of claim 36, wherein at least some of the lower
ends of the sample wells are respectively closed by single dialysis
membranes.
40. The apparatus of claim 36, wherein at least some of the lower
ends of the sample wells are closed by at least one common dialysis
membrane.
41. The apparatus of claim 36, wherein the dialysis membrane
comprises a plurality of membrane layers.
42. The apparatus of claim 36, wherein the dialysis membrane is
sprayed upon the sample plate.
43. The apparatus of claim 36, wherein the dialysis membrane is
adhesively connected to an underside of the sample plate.
44. The apparatus of claim 36, wherein the dialysis membrane is at
least one of bonded, welded, and sprayed on an underside of the
sample plate.
45. The apparatus of claim 36, wherein an adhesive foil is provided
as a releasable closure of the upper ends of the sample wells.
46. The apparatus of claim 36, wherein a cover is provided as a
releasable closure of the upper ends of the sample wells.
47. The apparatus of claim 36, wherein the dialysis container
comprises at least one inlet and at least one outlet, at least one
of which is connected to a float valve.
48. The apparatus of claim 47, further comprising means for removal
of substances to be dialyzed out of the dialysate, the removal
means in fluidic communication with the at least one outlet and the
at least one inlet.
49. The apparatus of claim 48, wherein the removal means comprises
at least one ion exchanger.
50. The apparatus of claim 48, wherein the removal means comprises
an absorber for binding detergents.
51. The apparatus of claim 48, wherein the removal means comprises
chelate forming substances.
52. The apparatus of claim 47, wherein dialysate flow through the
at least one inlet and through the at least one outlet is
adjustable.
53. The apparatus of claim 52, wherein the dialysate flow is
adjustable by at least one of a valve and a positioning
element.
54. The apparatus of claim 47, wherein the dialysis container,
including the at least one inlet and the at least one outlet, forms
at least a portion of a circulation system for the dialysate, the
circulation system further comprising a rotary pump.
55. The apparatus of claim 36, wherein the sample plate is held in
the dialysis container by at least one stand element disposed on a
bottom of the dialysis container.
56. The apparatus of claim 36, wherein the sample plate is held by
at least one or more float element disposed in the dialysate
contained in the dialysis container.
57. The apparatus of claim 36, wherein the means for holding the
sample plate includes means for providing movement and mixing of a
sample material contained in at least one of the sample wells.
58. The apparatus of claim 57, wherein the means for providing
movement and mixing comprises a shaker.
59. The apparatus of claim 57, wherein the means for providing
movement and mixing comprises an ultrasonic source.
60. The apparatus of claim 36 further comprising at least one
monitoring element for at least one of measuring and treating the
dialysate.
61. The apparatus of claim 60, wherein the at least one monitoring
element comprises means for simultaneous measurement of
conductivity.
62. The apparatus of claim 60, wherein the at least one monitoring
element comprises means for simultaneous measurement of optical
density.
63. The apparatus of claim 60, wherein the at least one monitoring
element comprises a fluorescence detector.
64. The apparatus of claim 60, wherein the at least one monitoring
element comprises a thermometer.
65. The apparatus of claim 60, wherein the at least one monitoring
element is provided for control of the dialysis process.
66. The apparatus of claim 36 further comprising means for
transferring a sample from the sample plate.
67. The apparatus of claim 66, wherein the means for transferring
the sample comprises a receiving plate, which can be inverted and
placed on the sample plate, the receiving plate including a
plurality of sample wells with a (n.times.8.times.12) matrix
arrangement corresponding to the matrix arrangement of the sample
wells of the sample plate.
68. The apparatus of claim 67, wherein the means for transferring
the sample comprises using centrifugation.
69. The apparatus of claim 67 wherein the sample wells of the
receiving plate define openings that correspondingly engage
openings defined by the sample wells of the sample plate to
establish a shape fit.
70. The apparatus of claim 69, wherein the sample wells of the
sample plate comprise a conical shape and the openings of the
sample wells of the sample plate are individually smaller than
corresponding openings of the sample wells of the receiving plate,
where the openings coincide when the receiving plate is set upon
the sample plate.
71. The apparatus of claim 69, wherein the openings of the sample
wells of the sample plate are individually substantially equal in
size to the openings of the sample wells of the receiving
plate.
72. The apparatus of claim 67, wherein the means for transferring a
sample further comprises a seal between the sample plate and the
receiving plate, the seal selected from at least one of a sealant
and a paste.
73. The apparatus of claim 36, wherein the means for the moving the
dialysate comprises a magnetic stirrer.
Description
DESCRIPTION
[0001] The invention concerns an apparatus for the simultaneous
dialysis of a plurality of liquid samples, which are contained in
sample wells in a sample plate, and have been brought to this
sample plate for a dialysis separation by at lest one semipermeable
membrane which is in contact with a dialysate. Such a dialysis
system can find general application where, for analytical purposes,
specifically a plurality of liquid microsamples is to be
investigated regarding their the macro-molecular apportionment and
from which microsamples, low molecular weight molecules, which
would disturb the analysis, must be separated. The separation is to
be carried out in an efficient, easily manipulated manner and at
the lowest possible expense. Beyond this, the dialysis system is to
be applied advantageously in order to concentrate macromolecular
containing microsamples quickly, protectively and without
substantial loss. A further area of application is the buffering of
samples, especially in the range of the DNA-treatment, for the
investigation of proteins and in the case of sequentially occurring
enzymatic reactions.
[0002] In recent years, highly parallel screening techniques, such
as High Throughput Screening=HTS and Ultra High Throughput
Screening=UHTS, have been showing up as analytical methods very
frequently in analytical work. This has been especially aided by
the efforts of the pharmaceutical industry for the capture of
targets for the development of newer pharmaceuticals. Also, the
proteomanic analysis is vigorously developing itself, and enables a
very high sample throughput for the characterization of a
multiplicity of proteins of a proteome with biological
modifications both in various conditions, such a healthy and ill.
Beyond this, the said proteomic analysis is an indispensable aid
for many application ranges in biochemical and biotechnical
research with high throughput analyses, for example, for the
characterization of enzymes in regard to their activity, for the
characterization of analytical and preparative chromatographic
separations, for the mass centered renaturation of protein samples
and for the characterization of nucleic acids.
[0003] These high throughput procedures have led, in their general
range, to acceptable auxiliary technologies for the development of
special analysis procedures. For example, these group themselves
around a microtiter of 8.times.12 analysis positions in the basic
area of a well plate of about 12.6.times.8.6 cm. At the present
time, attention is given to a trend for even further compression of
the analysis positions on the same basic area of
n.times.8.times.12, where n can equal 4, or even 16 and further to
more densely compacted multiple well plates. Very frequently, the
materials of interest can be macromolecular substances such as DNA
and its fractionates, proteins, peptides, glycoprotein and
synthetic macromolecules as well as combinations of these
substances.
[0004] Two additional and new procedures, MALDI-MS (Matrix Assisted
Laser Desorption Ionization Mass Spectrometry) and ESI-MS
(Electrospray Ionization Mass Spectrometry) have been recently
developed in the last few years, and have proved themselves as
excellent procedures for HTS/UHTS and for the proteomic analysis.
In this case, especially in combination with protease digestion
methods. In general, a strong tendency toward miniaturization of
the methods of analysis must be recognized. Many procedures for the
high-throughput screening method show a strong demand for sample
preparation with high requirements. Some of the requirements are
listed below:
[0005] 1. the samples may contain only a low concentration of salt
or detergent, or the samples must be found in a specific millieu of
ionization,
[0006] 2. the samples, which lie in the .mu.l-volume range, must be
treated in a highly parallelized manner, in order to guarantee the
required analysis frequency, The treatment must be carried out,
uniformly and under standardized conditions for all samples,
[0007] 3. the recovery for these microsamples in the process of the
sample preparation, must be satisfactorily high, and
[0008] 4. frequently, samples of biological material must be
protectively concentrated before the analysis, in order to achieve
the necessary desired level of concentration.
[0009] For the removal of low-molecular substances, for the
transfer of the samples into a defined millieu and for the
concentration of macromolecular materials, excellent results have
been acquired with the dialysis procedure requiring the use of semi
permeable membranes. Consequently, there has arisen a greater
number firms offering dialysis procedures, which, for example, can
be found collected together under the address of
http://biosupplynet.com.
[0010] The principal effort of the firms and the inventors for the
improvement of the dialysis technology, addresses those problems,
which relate to the practical manipulation of the technology. Some
of the problems to be overcome are: mixing, recovery of
macromolecules, difficulties with the speed of the dialysis
process, and operations which are connected with small volumes,
i.e., in the .mu.l-range.
[0011] Where the MALDI and the ESI-MS procedures are concerned, the
present state of the technology finds the removal of salts,
detergents and other contaminating substances necessary.
[0012] Possibilities, which could improve the quality of the
essential mixing of a dialysis solution, are described in U.S. Pat.
No. 5,183,564 and in U.S. Pat. No. 6,176,609. U.S. Pat. No.
6,176,609 in particular describes a general procedure for mixing in
a plurality of vessels. Giving consideration to the universal trend
for miniaturization, a series of solutions were suggested. Also,
U.S. Pat. No. 6,039,871 teaches of equipment which can be looked at
as being disposable and the dialysis of 10 to 100 .mu.l samples is
foreseen as being in special vessels which can be encapsulated, one
in the other. In U.S. Pat. No. 5,733,442 a closed microdialysis
system is proposed, which is marked by microchambers, which can be
screwed together, and possess two dialysis membranes and a special
stirring device.
[0013] The difficulties which arise in connection with recovery of
small analyte volumes is given attention by the inclusion of a
special capture chamber in U.S. Pat. No. 6,217,772.
[0014] In the documents U.S. Pat. No. 5,783,075 and U.S. Pat. No.
5,503,741 a proposal for floating dialysis in vessels of a special
configuration is made known. Where the floating dialysis is
concerned, there are also offerings from the firm PGC, the firm
Daigger and the firm Pierce, where the widely publicized slide
dialysis system is treated.
[0015] In order to increase the rapidity of the dialysis, giving
consideration to the ESI-MS technology, a capillary dialysis system
is taught by U.S. Pat. No. 5,954,959.
[0016] Where a multiplicity of samples must be treated, proposals
are put forth in U.S. Pat. No. 4,450,076. In these proposals the
dialysis vessel is placed about a central axle, and is turned by
means of rotation. Offers from PGC Scientifics Corp. bring forward
a central axis oriented, equilibrium chamber, which is sealed by
Teflon coated screws. Pierce Biotechnology, Inc. offers a
microdialysis system for 12.times.20-100 .mu.l volumes.
[0017] The proposed solutions of the problem, however, are not
adaptable to the demands of higher throughputs, such as are
necessary for HTS, UHTS and for proteomic analysis because of the
following, because they:
[0018] 1. are not acceptable for highly parallel microplate
technology especially where corresponding liquid treatment is
necessary,
[0019] 2. a simultaneous and essentially uniform dialysis executed
for all samples in a greater sample count in the .mu.l range is
prohibited,
[0020] 3. a satisfactorily sufficient recovery of these small
volumes fro secondary analytical procedures is not allowable,
[0021] 4. a correspondingly high dialysis speed is not
provided,
[0022] 5. the volumes required for the dialysate are inadequately
large, or
[0023] 6. because of their complicated manner of application, the
said solutions are not practical in routine operation with a high
throughput.
[0024] Thus, the invention has the purpose, of dialysing
simultaneously a multiplicity of micro samples in the .mu.l-range
essentially uniformly, wherein the manipulation is easily carried
out and at as low a cost as possible, whereby these dialyses can be
executed quickly and, if required, in an automatized manner, within
the requirements of the modem screen and analysis methods.
[0025] In the case of a large throughput of samples, the dialysis
permits a sufficiently high recovery of small volumes for secondary
analytical procedures.
[0026] In accord with the purpose of the present invention, there
is proposed an apparatus for the simultaneous dialysis of a
plurality of liquid samples, wherein:
[0027] there is included a dialysis vessel with a dialysate as well
as means for the inflow and outflow of the dialysate, which remain
in connection with a level control,
[0028] there is included at least one sample plate, which is either
immersed in, or is in contact with, the surface of the dialysate,
as well as being held by holding elements, in or on the surface of
the dialysate, with a matrix (n.times.8.times.12) arrangement of a
plurality of equal sample wells, which arrangement is acceptable
and known for liquid handling technique in microplate technology
and said wells have holding capacities for microliter volumes,
whereby, of the sample wells, respectively, the upper ends are open
and the lower ends are closed by a semipermeable membrane lying in
a plane, and the sample plate in the area of the said matrix of the
matrix of the sample wells has no gas barrier forming or supporting
zones or elements which extend beyond the plane of the
semipermeable membrane and the said sample plate 3 has, in its rim
areas, elements, such as openings for the release of air imprisoned
by touching contact with the dialysate,
[0029] and besides the above, the said apparatus contains means for
the movement of the plate and the dialysate.
[0030] Enabled by the acceptability of the sample plate used in the
dialysis system, for the said liquid handling technique for the
microplate technology of liquid, samples can be prepared with a
high degree of throughput in accord with this technology, then
subsequently be employed for their purpose and finally recovered
for further use. With the special design of the sample plate, those
air barriers which obstruct the dialysis upon the immersion of, or
the displacement of the plate into or onto the dialysate surface
are avoided. Should procedurally evolved gases migrate into the
contact zone between the dialysate and the sample plate, then these
can be forced out by deaerating apparatuses, so that in any case,
an unbroken contact can be assured between the dialysate and the
sample plate without the said gas barriers, such as air bubbles and
the like. This disturbance-free contact is an essential
presupposition for simultaneous and essentially uniform dialysis
for each of the large number of samples in the .mu.l range. This
effect is essentially supported by the movement of the sample
liquids and the dialysate, so that, predominately, no secondary
membrane formation between the dialysate and the sample volumes can
form and the dialysis can continue with unbroken continuity. To
serve this purpose, the dialysis vessel possesses at least one
entry and one exit opening, in order that the materials which are
accumulated in the dialysate can again be continuously expelled
from the said dialysate and continually a dialysate with equal
dialysis acceptance power remains available in the system. The
entry and exit flows are, meanwhile, connected by a level control,
in order to hold constant the conditions of dialysis on the
semipermeable contact between the sample liquid and the dialysate,
thus maintaining the above advantages with consideration for level
control.
[0031] By means of the movement of not only the dialysate, but also
of the sample liquid, this movement being done by a known shaking
device, with which the sample plate is connected, not only is the
high dialysis effectivity itself attained, but also special usages,
notably dialysis-effectivity is achieved. Of the latter, a dialysis
of detergents which form micellla, is already enabled.
[0032] With the above stated features, the realization of variously
designed dialysis systems is enabled, wherein the said dialysis
systems can carry out different applications, either manually or
with automatic drive, these being independent therefrom, as to
whether the sample plate, for instance, by means of a pivot or a
shaking arm lies on the dialysis vessel or floats in the dialysate
which is present in the container.
[0033] In the most simple case, without limiting the invention, the
sample plate may consist of a plate with cylindrical recesses or
wells in the receiving means for microliter volumes. On the
underside of the sample plate are the borings (sample containing
recesses) either respectively closed by a common or by individual
dialysis membranes, which, for example, are adhesively held on the
underside of the plate. They may also be welded, bonded, or sprayed
on. The dialysis membranes can also consist of more than one
layer.
[0034] A cover or an adhesive film of a releasable closure of the
upper end of the sample vessel protects the sample material which
is in the sample wells, and blocks any evaporation and
contamination of the small quantity of sample in the microliter
range.
[0035] Described and explained in the subordinate claims, are a
multitude of advantageous embodiments of the invented features. In
this way, the dialysis vessel with the accepted sample plate
becomes an integral part of a circulation system. In such a
circulation system, for example, with a pump controlled, recycling
apparatus, it is possible that an ion-exchange device or a
detergent capturing adsorber could be placed. Such an addition
would hold the concentration of the substances which are to be
removed from the dialysate to a very small level, and thereby the
speed of the dialysis procedure would be increased and the
necessary volumes of the dialysate would be simultaneously
minimized. Also the use of bound substances which form complex
substances is possible, in order to remove metal ions.
[0036] In the following, the invention is described and explained
with the aid of embodiments as shown in the drawing. There is shown
in:
[0037] FIG. 1: A sample plate, secured at the base of a dialysis
vessel by feet,
[0038] FIG. 2: An apparatus with a floating holder of the sample
plate on the surface of the dialysate,
[0039] FIG. 3: A dialysis vessel with the holder of the sample
plate in accord with FIG. 1, with both entry and exit fittings for
the dialysate.
[0040] FIG. 4: A dialysis vessel with a sample plate in a
circulation system for the removal of interfering substances from
the dialysate,
[0041] FIG. 5: An apparatus for dialysis, wherein the sample plate
is held in a shaking device to create turbulence in said dialysis
apparatus,
[0042] FIG. 6: A floating holding means for the sample plate (see
FIG. 2) with conical sample wells,
[0043] FIG. 7: A sequential run of a procedure to transfer dialyzed
sample material subjected to centrifugation out of the sample plate
with conical sample wells into a receiving plate with cylindrical
wells,
[0044] FIG. 8: A second sequential run of a procedure similar to
FIG. 7, showing a sealing means between the sample plate and the
receiving plate, and
[0045] FIG. 9: A graph showing the conductivity of the dialysate
during a period of dialysis.
[0046] In FIG. 1 is shown a dialysis container 1 with a dialysate 2
therein. On the bottom of, and within the dialysis container, is to
be found a sample plate 3, which is secured by a holder 4. This
sample plate 3 consists of a plate shaped, basic body in which, and
within the specifications of a known liquid handling technique for
acceptable microplate technology, is placed an 8.times.12 matrix of
aligned cylindrical wells 5 for the acceptance of sample material
6, the content of each well being in the microliter range.
[0047] On the underside of the sample late 3 is found a dialysis
membrane 7, this membrane being semipermeable, common to all sample
wells and secured on the rims thereof by adhesive. By means of this
dialysis membrane 7, each individual portion of the sample material
6, which is within the wells 5, stands respectively in contact with
the dialysate 2. For this purpose, the sample plate 3 is so
supported by the holder 4, that the said plate 4 is immersed, with
its dialysis membrane 7, into the dialysate. By means of the
dialysis membrane 7, the exchange of small molecules is possible,
in accord with the exclusion threshold, since a concentration
equilibrium between the dialysate 2 and the liquids of the sample
material 6 is in force. The removal of the said small molecules out
of the material 6 of the samples is accomplished by the effort of
the said solutions to establish the mentioned equilibrium between
the two compartments. Large molecules are restrained from passing
through the dialysis membrane 7.
[0048] The plane of the dialysis membrane 7 incorporates, in a way,
also the lowest level of the sample plate 3, during its operation
in accord with its application. There exists in this matrix area of
the sample wells 5 no zones or elements for stabilization
fastening, manipulation or the like, or even yet areas dependent
upon fabrication, which would protrude from the sample plate 3
outward and beyond, which would contactingly impinge on the
dialysate 2, or be immersed therein. Further the arrangement is
such that no extension of the said zones or elements exist, which
would interfere with the uniformly running dialysis process by
introducing air or creating air barriers.
[0049] By means of a known magnetic stirrer 8 on the bottom of the
dialysis container 1, the dialysate is held in motion, in order
that a concentration gradient on the dialysis membrane 7 be held as
small as possible and also to accelerate the dialysis. An adhesive
foil 9, serving as a releasable closure of the upper rims of the
sample wells 5, protects the sample material 6 which is found
therein and prevents an evaporation or a contamination of the
sample very small volumes.
[0050] FIG. 2 depicts a construction, which is very similar to that
of FIG. 1. The difference, in this case, is that the sample plate 3
is not supported by foot or structural holding elements 4 rigidly
connected to the bottom of the dialysis container 1, but is held by
means of a framelike, float element 10 directly on the surface of
the dialysate. This is accomplished in such a manner, that the
dialysis membrane 7 lies on this surface. This mode of holding is
independent of the level of the dialysate 2 in the dialysis
container 1.
[0051] Additionally, the sample plate 3 has air escape openings 11,
which allow gas collecting under the sample plate 3 to bleed out,
thereby assuring an unbroken contact of the dialysis membrane 7
with the surface of the dialysate 2. This complete contact coverage
forms the necessary preparation for a simultaneous and essentially
uniformly completed dialysis for each sample of this large number
of samples in the .mu.l-volume range.
[0052] Because the sample plate 3, at least in the matrix area of
the sample wells 5, possesses no elements (for holding or the
like), which would protrude downward beneath the plane of the
dialysis membrane 7 and thus immerse themselves in the dialysate 2
or cause turbulence in the same, either of which would disturb the
desired uniformity of simultaneous analyses, very quickly essential
characteristics for value-determining usage of the sample plate 3
in a dialysis system were immediately taken advantage of, in order
to avoid air locks, or at least not to support them, in the contact
zone of the dialysate 2 against the sample plate 3. If,
nevertheless, gases evolved from processing appeared in this zone,
then, these gases, as mentioned above, could disperse through the
air escape openings 11 and emerge above trough the sample plate 3.
Instead of the air escape openings 11, other gas dispersing
elements were given consideration, such as edge phase-changing, or
the like. To enhance clarity, details of the air escape openings 11
(or other deaeration equipment) were not explicitly shown in each
figure presentation.
[0053] FIG. 3 shows an apparatus for dialysis, wherein the sample
plate 3 (as in FIG. 1) is supported in the interior of the dialysis
container 1 on the floor thereof by means of feet or standard
holding devices 4. The dialysis container 1 possesses in this case,
a feed fitting 12 as well as a outlet fitting 13 for the dialysate
2. The level of the dialysate 2 is regulated by means of an
adjustable float 14 with a float actuated valve 15. In this case,
the float 14 is guided to be vertically movable in a float track
16. The advantage of this, is to be found in the continuous content
balancing of the dialysate 2. In this way, the adjustment of a
concentration equilibrium between the dialysate 2 and the
respective sample material 6 contained in the wells 5 can be
avoided, also the speed of the dialysis is greater and the removal
of low molecular substances from the sample material 6 is
fundamentally improved. Naturally, the float valve system (12-17)
can be replaced by an electronic level controller which regulates
the feed at inlet 12.
[0054] FIG. 4 shows an apparatus for dialysis, wherein the dialysis
container 1, which is shown in top view, demonstrates the therein
placed sample plate 3 (see FIGS. 1 and 3), and shows the inlet 12
as well as the outlet 13 as being components of the circulation
system for the through-flow of the dialysate 2. The exchange of the
dialysate 2 not carried out, as in the apparatus of FIG. 3, through
an open system, but rather by the dialysate 2 being transported by
a pump 17, through a filter cartridge 18 and into a line 19, with
the circulation system being completed by passage through the
dialysis container 1. The direction of the dialysate 2 is indicated
by an arrow, whereby the black arrows symbolize the exit flow of
the dialysate 2. Upon its exit out of the dialysis container 1, the
dialysate 2, for example, can be enriched with ions and/or
detergents from the sample material 6. By means of one or more
sorbents in the filter cartridge 18, these components can be
removed from circulation. The cleaned dialysate 2 thus migrates, as
the white arrows show, back into the dialysis container 1.
Advantageously, here, the ubiquitous applicability of the dialysate
2 contributes to:
[0055] (a) the said avoidance of the adjustment of a concentration
equilibrium, between the dialysate 2 and the sample material 6 held
respectively in the sample wells 5 and
[0056] (b) the thereto connected advantages for dialysis (see the
embodiment example of FIG. 3).
[0057] FIG. 5 depicts an apparatus for dialysis (once again
sectional profile and top views), wherein the sample plate 3 is
neither anchored to the bottom of the dialysis container 1 (see
FIG. 1) nor is it floating on the dialysate 2 surface in the said
dialysis container 1 (see FIG. 2). Rather the sample plate 3 is
held by a holder 20, which is also connected to a shaking device 22
through a shaker arm 21. The shaker 22 serves, as the white,
crossed arrows indicate, for the horizontal movement of the sample
plate 3 along the surface of the dialysate 2 and also moves the
sample plate 3 within the amplitude and frequency limits as
directed for a shaker installed for microtiter sample plates as
these limits are defined for laboratory operation. In this way, the
sample material 6 found in the sample wells 5 of the sample plate 3
is thoroughly mixed, which acts against the establishment of
concentration gradients in the said sample material 6, as well as
in the dialysate 2, which the shaker 8 also affects. This has the
favorable advantage, that the somewhat hindering construction of
secondary membranes, which are necessary for many dialysis
processes, may be omitted. Also, the transporting away of gas
bubbles in the area of the membrane is favored by this shaking
motion.
[0058] The advantage of this apparatus is, that not only is the
recirculation and cleaning associated with the content balance of
the dialysate 2, as is described for the embodiments of FIG. 3, 4,
omitted, but also the speed and completeness of the dialysis is
improved. To preserve clarity, a combined presentation with the
said, and previously described embodiments, is not specifically
illustrated in the attached drawings. In an additional embodiment
example, not shown here, the motion of the samples and dialysate
can be carried out with the same positive effects also by being
coupled with an ultrasonic mixer.
[0059] FIG. 6 shows (likewise in sectional profile and top views)
an apparatus for dialysis, wherein the sample plate 3, as is the
case in FIG. 2, floats on the surface of the dialysate 2. The
sample plate 3 does not possess, as was the case in the previously
described embodiments, sample wells with cylindrically parallel
walls, but sample wells 5a which are conically tapered, in the form
of a cone frustum with respectively larger lower openings, which
are closed by the dialysis membrane 7. The said wells 5a have, in
comparison to the lower openings, smaller upper openings. The
smaller upper openings create an advantageous shape-closure (see
FIG. 7) for the transfer of the sample material 6 after the
dialysis (following the dialysis) into the individual well volumes,
in the same matrix in another sample receiver plate. This procedure
is in accord with known microtiter plate technology. Easily
recognizable in the top view presentation of FIG. 6 is adhesive
foil 9 over the matrix arrangement of the sample wells 5a, which
foil prevents evaporation, spilling, and contamination of the
sample material 6, which is in the wells 5a during the dialysis or
can occur even during transport.
[0060] FIGS. 7, 8 show, schematically, respectively in sectional
views through the plate, a sequential run of the procedure through
centrifugation, out of the sample plate 3 with conical sample wells
(see FIG. 6) into a receiver plate 23 with cylindrical wells. The
receiving plate 23, in an upset position, is placed on top of the
sample plate 3, whereupon the two are turned over in common. By
means of centrifugation, the sample material 6, which is originally
in the sample plate 3 is transferred to the receiver plate 23.
Subsequently, the sample plate 3 and the receiver plate 23 are
taken apart. As a centrifugal device, the known laboratory
centrifuge for microtiter plates can be used. In FIG. 7, the upper
openings of the wells 5 are smaller than the openings of the sample
container of the receiver plate 23 which confronts them. On this
account, the already described satisfactory shape-fit assures the
penetrative interconnection as shown in the drawing 7. Conversely,
in FIG. 8, we see the confronting well openings of the sample plate
3 and the receiving plate 23 respectively equal in size. In this
case, a required tighter shape fit is assured by means of an
intermediately inserted sealing means 24 between the sample plate 3
and the sample plate 23.
[0061] In the following four embodiments is shown, how, with the
described apparatuses, different substances can be dialyzed.
APPLICATION EXAMPLE NO. 1
[0062] Low-molecular substances in 96 samples such as p-nitrophenol
(p-NP) and sodium chloride, are to be uniformly removed, in short
dialysis periods, wherein the reception of the sample plate 3 in a
shaking device (see FIG. 5) is provided and which said plate 3, in
accord with FIG. 4, is connected into a circulating system for the
dialysate 2.
[0063] For this situation, into the 8.times.12 dialysis wells,
which are closed with a VSMP Millipore membrane (0.025 .mu.m), each
100 .mu.l of a 1.5 mM p-NP solution in 50-mM diethanol-amine buffer
pH=9.8 (DEA), which, in addition, contains 750 mM NaCl, is pipetted
and dialyzed against a volume, which is only 11 times greater than
a volume of 110 ml deionized water for 2 hours. The dialysate 2 is
circulated by a hose pump. In the circuit is integrated a
deionizing column (Eco Pac, 10 ml, Bio Rad) (see FIG. 4). During
the dialysis, the sample plate 3 is continually shaken, which
sample plate is closed with adhesive foil 9 and is fastened in the
holder 20 of the shaping apparatus (see FIG. 5). The effectivity of
the separation of the lower molecular nitrophenols following the
dialysis is checked with the absorbencies of the outlet solution.
The absorbency measurement of the outlet solution, which emerges
from the dialysis, is carried out with a DEA-buffer solution,
pH=9.8 in a microtiter plate and the absorbencies read off on a
display. For the absorbency measurement of the 96 dialyzed samples,
3 aliquots are taken by pipette from the sample plate 3, with 50 mM
DEA-buffer solution pH=9.8, mixed in a microtiter plate and
measured in a display.
[0064] The comparison of the absorbency in the 96 positions of the
dialysis module is shown in Table 1. The measured absorbencies have
been reduced by the blind buffer value.
1TABLE 1 Comparison of the Absorbencies at 405 nm (A.sub.405)
before and after Dialysis A.sub.405 Analysis Dilution Solution
Factor Av. Val. Analysis Samples (n = 96) Solution A.sub.405 Total
p-NP(%) p-NP pre Dialysis 0.397 .+-. 0.005 30 11.910 100 p-NP post
Dialysis 0.028 .+-. 0.002 3.75 0.105 0.88
[0065] The values indicate, that under the described conditions,
more than 99% of the p-NP can be removed. The distribution of the
absorbency values after the dialysis show, that the dialysis speed
in all 96 dialysis wells is very much the same. Besides the
comparison of the absorbencies before and after the dialysis, the
conductivities of the employed samples were measured and were
compared with the conductivities after the dialysis of the 96
dialysis wells. From these values a residual capability of
conductivity was determined, in relation to the outlet solution of
0.2%, which, in any case, confirms the effectivity of the
dialysis.
APPLICATION EXAMPLE NO. 2
[0066] For the removal of lower molecular ions from proteins, in
8.times.11 positions of the sample plate 3, per well, 75 .mu.l of
concentrated solution of an alkaline phosphatase (14.5 .mu.g/ml)
was added to IM DEA buffer pH=9.8, and dialyzed for one hour
against a large volume of 470 ml deionized water. The sample plate
3, which is placed as a floating element 10 on a polysterol
framing, and floats on the dialysate 2, which is kept in motion by
the magnetic stirrer 8 (see FIG. 2). The continuous removal of the
low molecular substances in the sample wells 5 is monitored by the
measurement of the conductivity (see FIG. 9) in the said dialysate
2.
[0067] After 60 minutes of dialyzation, 60% of the ions which can
be dialyzed have been removed. The determination of the enzymatic
activities of the alkaline phosphatase in the 88 occupied
dialyzation wells of the sample plate 3 and a comparison of the
enzymatic activity with the outlet solution yielded a recovery of
the enzymatic activity of 90.4+4.3%.
APPLICATION EXAMPLE NO. 3
[0068] On the example of Triton X-100 (TX-100), the point is to
determine, if it is possible to remove this much used detergent by
dialysis from analysis samples. To this end the sample plate 3 is,
respective by wells, charged with 7511 of a 0.5% aqueous solution
of Triton X-100 and dialyzed for 8 hours vs tap water. The sample
plate 3 was closed by adhesive foil 9, and again placed in the
holder 20 of the shaker apparatus (see FIG. 5) for continuous
agitation. During this part of the operation, the same was immersed
in 170 ml of tap water as a dialysate 2, which was renewed at flow
rate of 170 Ml/min in a circulation system in accord with FIG.
4.
[0069] In order to capture eventual volume changes, the sample
plate 3 was weighed before and after the dialysis. For the
determination of the effectivity of the removal of the Triton by
dialysis, aliquots were taken by multipipettes from the dialysis
containers of the module, mixed in a microtiter plate with 30%
n-propanol and measured in a fluorescence display at an excitation
wave length of 270 nm from an emission wave length of 310 nm. The
Triton X-100 which was subjected to dialyzation, after dilution in
30% n-propanol was measured under the same conditions in a
microtiter plate. For the correction of the measured values and for
the regulation of the linearity of the range of measurement, both
the propanol solution in 56 positions of the microtiter plate
(blind values) and three standard concentrations of Triton X-100,
between 12.5 and 50 .mu.M under the same conditions were measured.
The results are summarized in Table 2. The measured average values
were reduced by the determined propanol blind value:
2 Dilution Fluorescent Factor Analysis Solution Analysis
Fluorescence Samples Av. Val. (n = 96) Solution Total TX-100 (%)
TX-100 739.6 .+-. 11.83 201 148 660 100 pre Dialysis TX-100 62.39
.+-. 6.24 30 1 872 1.26 post Dialysis
[0070] The measured fluorescences following the dialysis in the 96
dialysis wells of the sample plate 3 show, that under the described
conditions, 98.7% of the Triton X-100 was uniformly removed from
all 96 of the dialysis wells.
APPLICATION EXAMPLE NO. 4
[0071] The sample plate 3 can also be employed for the simultaneous
concentration of 96 samples. For this purpose, in accord with each
well, 100 .mu.l of a 0.3% Dextra-blueing solution with a
multi-pipette was placed in the sample wells 5 of the sample plate
3. The sample plate 3 was subsequently fixed on the floating frames
10 (see FIG. 2) of polysterol, which were laid on 100 ml of a 30%
aqueous polyethylene glycol solution (PEG 40 000). After 45
minutes, the volume reduction was quantitatively determined. To
this, from 88 out of the 96 positions was taken, per position, 30
.mu.l with a multi-pipette and transferred into a microtiter plate,
which, per container, contained 120 .mu.l 50 mM DEA-buffer
solution. In the remaining 8 positions of the microtiter plate,
instead of the sample solution, each was given 30 .mu.l reference
solution (0.3% Dextra-blueing solution). The microtiter plate was
then, after intensive mixing, measured in a display at 620 nm. A
comparison of the determined absorbency of samples and reference
solution shows, under the described conditions, the measured
absorbency.
3TABLE 3 Comparison of the Absorbency of Dextra Blueing before and
after concentration Samples A.sub.620 Reference Solution 0.291 .+-.
0.012 (n = 8) Samples after Concentration 0.465 .+-. 0.020 (n =
88)
[0072] The absorbencies have increased by a factor of 1.6. That
means, that after 45 minutes the volume of the samples is reduced
by 37.5 .mu.l, and indeed relatively uniformly, as may be seen by
the distribution.
APPLICATION EXAMPLE NO. 5
[0073] For the dialysis of plasmid DNA, in a 96 well
dialysis-plate, 110 .mu.l of the samples having plasmid DNA pc
DNA3.1hcS.DELTA.E44-N63(6.5 kb) was treated against buffer Tris 10
mM pH 8 and subjected to shaking and mixing. To the samples,
paranitrophenol (PNP) was added in the end concentration of 978
.mu.M and the reduction of the concentration was measured
respectively after two and four hours.
[0074] The content of DNA in the samples was likewise determined
after two and four hours in, respectively, eight parallel samples
of a concentration with the aid of optical density. The detection
was carried out at 400 nm for PNP and 260 nm for the plasmid
DNA.
[0075] The table below shows the balance of the plasmid-DNA: That
is, the concentrations were computed with a standard series of
plasmids by optical density.
[0076] Table:
4 After 4-hour Output DNA Dialyzation, DNA in .mu.g/ml in .mu.g/ml
5 4.55 10 9.32 50 50.79
[0077] The balance of PNP in the samples, as per a standard series
for the samples where n=32:
5 PNP after 2 hour PNP after 4 hour Item Output PNP Dialysis
Dialysis Concentration in the 978.6 38.96 0.56 sample in .mu.M
Standard deviation 1.01 4.6 0.60 in .mu.M % from output 100 3.9
0.06
[0078] Reference Numbers and Corresponding Components
[0079] 1 Dialysis container
[0080] 2 Dialysate
[0081] 3 Sample plate
[0082] 4 Holder
[0083] 5 Well, (5a conical tapered well)
[0084] 6 Material of the sample
[0085] 7 Dialysis membrane
[0086] 8 Magnetic stirrer
[0087] 9 Adhesive foil
[0088] 10 Float element
[0089] 11 Air escape opening
[0090] 12 Entry fitting (feed)
[0091] 13 Outlet fitting (exit)
[0092] 14 Float
[0093] 15 Float valve
[0094] 16 Float guide
[0095] 17 Pump
[0096] 18 Filter cartridge
[0097] 19 Line, for circulation of liquid
[0098] 20 Holder (FIG. 5)
[0099] 21 Shaker arm
[0100] 22 Shaker
[0101] 23 Receiving plate (receives contents of 3)
[0102] 24 Sealing means
* * * * *
References